Studies analyzing the specificity of receptors on T cells and their ability to distinguish between structurally related antigens in comparison to that of humoral antibody has provided insight into the nature of the T cell receptor. Reports assessing the angigen specificity of cytotoxic T cells has indicated that such cells sensitized with one hapten cross react to a minor extent when tested against a similar hapten(s). I tested the specificity of cytotoxic T cells sensitized to the haptens TNP and dinitrophenyl (DNP). Since these molecules are structurally very similar and couple to proteins by attaching primarily to the same amino acid residues, the fine specificity of the receptor on the cytotoxic T cell and its ability to discriminate between closely related antigens could be determined. I observed that spleen cells sensitized against TNP modified stimulator cells displayed a cytotoxic effect against syngeneic TNP modified but not DNP modified target cells. The same finding was observed in the opposite direction. That is, effector cells sensitized against DNP modified cells did not cross kill TNP modified targets. The specificity of the anti-TNP effector cells was confirmed in a cold target competition assay. Presensitization in vivo with hapten modified cells followed by rechallenge in vitro did not alter the specificity of the response between the haptens. These data indicate that the receptor(s) on the cytotoxic T cell can distinguish between two very closely related haptenic molecules.